SOME  OBSERVATIONS  ON  METHODS  FOR  THE  DETEC- 
TION OF  B.  COL1  COMMUNIS  IN  WATER. 


By  E.  E.  IRONS,  Chicago. 


Reprinted  from  the  Proceedings  of  the  Twenty-Eighth  Annual  Meeting , held  at 
Indianapolis , Indiana , October  22,  23,  24,  25  and  26,  1900. 


COLUMBUS,  OHIO: 

THE  BERLIN  PRINTING  COMPANY. 
1901. 


I m 


SOME  OBSERVATIONS  ON  METHODS  FOR  THE  DETEC- 
TION OF  B.  COLI  COMMUNIS  IN  WATER. 

By  E.  E.  IRONS,  Chicago. 

In  the  course  of  an  extended  examination  of  polluted  river  waters 
for  the  Sanitary  District  of  Chicago,  a number  of  methods  for  the 
detection  of  B.  coli  communis  have  been  regularly  employed,  and  still 
others  experimented  with.  Four  methods  have  been  especially  stud- 
ied : (1)  the  dextrose  fermentation  tube,  (2)  the  lactose  fermenta- 
tion tube,  (3)  the  carbol-broth  method,  (4)  the  lactose  plate. 

For  the  dextrose  and  lactose  fermentation  tubes,  broth  was  pre- 
pared from  fresh  meat  from  which  the  muscle-sugar  had  been  ex- 
tracted by  Smith’s  method.  One  per  cent,  of  sugar  was  then  added 
to  the  broth.  The  initial  reaction  was  made  neutral  to  phenol- 
phthalein,  experiments  having  shown  no  appreciable  difference  be- 
tween the  results  with  this  reaction  and  others  slightly  more  acid.  Ex- 
periments likewise  showed  no  material  difference  between  tubes  con- 
taining 1 per  cent,  and  2 per  cent,  of  sugar.  Other  factors  of  varia- 
tion, over  which  we  have  no  control,  probably  outweigh  these  smaller 
differences.  The  cultures  were  incubated  at  38°  C.  for  48  hours,  gas 
readings  being  taken  after  24  hours  and  48  hours.  No  material 
changes  were  observed  if  .the  incubation  period  was  prolonged  to 
three  days.  Tubes  showing  gas  formation  were  removed  from  the 
thermostat,  cooled  to  the  temperature  of  the  room  and  the  absorption 
of  CO2  determined  by  the  addition  of  a 2 per  cent,  solution  of  NaOH. 
Sometimes,  particularly  in  the  larger  tubes,  the  absorption  was  in- 
complete after  the  first  addition  of  NaOH,  and  more  had  to  be  added. 

Carbol-broth  was  made  by  adding  1 c.c.  of  a 1 per  cent,  solution  of 
carbolic  acid  in  sterile  water  to  tubes  containing  9 c.c.  of  sterile  com- 
mon broth1.  From  such  tubes,  incubated  24  hours  at  38°  C.,  lactose 
plates  were  inoculated  and  placed  in  the  thermostat  for  24  hours. 
Fish  cultures  from  resulting  red  colonies  were  made,  and  the  growth 
reactions  determined  in  milk,  gelatin,  potato,  sugar-free  broth  for 
indol,  and  the  dextrose  fermentation  tube. 

Litmus-lactose-agar  was  prepared  by  adding  to  sugar-free  agar,  of 
a reaction  5 alkaline  to  phenolphthalein,  1 per  cent,  lactose  and 
enough  blue  litmus  solution  to  give  a clear  blue  color.  Plating  with 
the  water  direct  was  carried  on  in  the  usual  manner.  The  plates  were 
incubated  at  38°  C.  for  24  hours. 

1 Except  where  otherwise  stated,  media  were  prepared  according  to  the  “Procedures  Re- 
commended for  the  Study  of  Bacteria.”  Joiirn.  Anier.  Pub.  Health  Association.,  Jan.,  1898 


DETECTION  OF  B.  COLI  COMMUNIS. 


2 


THE  FERMENTATION  TUBE. 

Approximately  1100  determinations  were  made  by  the  fermentation 
tube  with  dextrose  and  lactose.  Lactose  was  used  in  hope  that  the 
trouble  caused  by  B.  cloacae  might  be  avoided,  since  B.  cloacae  fer- 
ments lactose  very  slowly  as  compared  with  B.  coli,  but  as  will  ap- 
pear presently  this  expectation  was  not  justified.  The  results  obtained 
with  dextrose  broth  seem  to  warrant  the  assumption  that  when  the  gas 
formula  shows  a marked  excess  of  H over  CO2  (i.  e.,  approximately 
a 2-1  formula)  B.  coli  is  generally  present.  A number  of  tubes  giving 
such  a formula  were  examined,  and  only  rarely  did  we  fail  to  isolate 
an  organism  closely  approximating  typical  B.  coli.  Moreover  when 
failure  to  isolate  B.  coli  did  occur,  B.  coli  may  still  have  been  present, 
but  overlooked  among  the  large  number  of  other  colonies  on  the 
plate.  Experiments  with  artificially  mixed  cultures  of  B.  coli  and 
B.  cloacae,  freshly  isolated  from  water,  showed  sometimes  a coli, 
sometimes  a cloacae  gas  formula,  but  in  the  majority  of  cases  B.  coli 
gained  the  upper  hand  and  an  excess  of  H appeared  in  the  tubes. 
When  the  fermentation  tube  is  used  for  highly  polluted  waters,  it 
occasionally  happens  that  there  is  little  or  no  formation  of  gas  even 
though  B.  coli  is  probably  present  as  shown  by  the  typical  gas 
formula  in  higher  dilutions  of  the  same  water.  This  difficulty,  how- 
ever, may  be  obviated  in  large  measure  by  using  several  dilutions 
where  the  pollution  of  the  water  is  not  approximately  known. 

The  following  table  (Table  A)  gives  the  totals  obtained  in  a series 
of  59  determinations  in  duplicate  with  dextrose  and  lactose  broth. 
Each  determination  with  dextrose  was  made  simultaneously  with  the 
same  dilution  of  the  water  as  the  corresponding  lactose  determination, 
and  both  were  kept  as  nearly  as  possible  under  identical  conditions. 
Both  polluted  and  comparatively  pure  river  waters  were  employed  in 
this  series. 


TABLE  A. 


Dextrose  Fermentation  Tube. 

Lactoio 

Fermentation  Tube. 

+ 

— 

? 

+ 

— 

? 

27 

19 

13 

21 

29 

9 

+ = total  gas  more  than  20%;  formula  showing  marked  excess  of  H. 

— = no  gas;  gas  less  than  10%. 

? = total  gas  10%-20%;  total  gas  more  than  20%  with  formula  showing 
excess  of  CO2. 


DETECTION  OF  B.  CO  LI  COMMUNIS. 


3 


The  table  shows  that  for  general  work  dextrose  is  to  be  preferred 
to  lactose.  This  is  true  for  both  polluted  and  unpolluted  waters  as 
is  shown  if  the  results  obtained  from  the  two  classes  of  waters  are 
tabulated  separately.  It  is  of  course  recognized  that  the  B.  enteri- 
tidis  group  ferments  dextrose,  but  not  lactose.  However,  germs  of 
this  class  are  probably  rare  in  river  waters. 

THE  CARBOL-BROTH  METHOD. 

Several  hundred  determinations  were  made  by  the  use  of  carbol- 
broth  followed  by  the  lactose  plate.  The  following  table  (Table  B) 
shows  the  totals  obtained  in  a series  of  duplicate  tests  made  to  ascer- 
tain the  comparative  value  of  the  dextrose  fermentation  tube  and  the 
carbol-broth  method  on  both  polluted  and  relatively  unpolluted  river 
waters. 


TABLE  B. 


Dextrose  Fermentation 
Tube. 

Carbol-broth  Method. 

_L 

— 

? 

+ 

— 

? 

Polluted  Waters 

33 

31 

5 

38 

30 

1 

Relatively  Unpolluted 
Waters 

56 

38 

25 

37 

61 

21 

Dextrose  fermentation  tube:  -f-,  — , ?,  as  in  Table  A. 

Carbol-broth  method: 

+ = red  colonies  on  the  lactose  plate,  which  give  the  typical  reactions 
of  B.  coli  in  (1)  milk,  (2)  gelatine,  (3)  potato,  (4)  sugar-free  broth  for  indol, 
(5)  the  dextrose  fermentation  tube. 

— — no  red  colonies;  red  colonies  do  not  give  an  excess  of  H in  the 
dextrose  fermentation  tube,  or  they  fail  in  the  other  reactions. 

? = red  colonies  fail  to  give  one  or  two  of  the  first  four  typical  reac- 
tions above.  . 

From  the  results  in  Table  B,  it  is  apparent  that  the  carbol-broth 
method  gives  a larger  number  of  positive  determinations  for  B.  coli 
than  does  the  dextrose  fermentation  tube,  when  polluted  waters  are 
tested.  On  the  other  hand,  with  relatively  pure  waters,  the  dextrose 
tube  is  shown  to  be  a much  more  delicate  test  than  the  carbol-broth 
method. 

THE  LACTOSE  PLATE. 

Comparative  experiments  on  the  use  of  the  lactose  plate  made  in 
series  with  the  dextrose  fermentation  tube  as  a control,  resulted 
rather  unfavorably  for  this  method.  In  many  cases  the  plates  made 
with  lactose  agar  having  a reaction  of  5 alkaline  to  phenolphthalein, 


4 


DETECTION  OF  B.  COLI  COMMUNIS, 


failed  to  show  any  red  colonies,  when  the  dextrose  tube  gave  a 2-1 
gas  formula.  When  the  alkalinity  was  reduced  to  5 acid  to  phenolph- 
thalein,  many  red  colonies  appeared  which  gave  either  an  inverted 
gas  formula,  or,  more  often,  no  gas  at  all  with  dextrose,  although  the 
control  dextrose  tube  showed  the  probable  presence  of  B.  coli. 

In  polluted  waters  where  B.  coli  is  present  together  with  other 
slightly  acid  forming  species,  the  excess  of  the  acid  of  B.  coli  may  so 
far  neutralize  the  alkalinity  of  the  medium  that  the  weaker  acid  pro- 
ducers appear  as  red  colonies.  Hence  there  is  a possibility  of  error 
in  assuming  that  the  number  of  red  colonies  on  a plate  indicates  the 
presence  of  a proportionate  amount  of  B.  coli. 

In  general,  results  show  that  where  the  waters  to  be  examined  are 
much  polluted,  or  where  the  amount  of  B.  coli  is  small  and  the  colony 
count  large,  the  lactose  plate  for  plating  water  direct  is  inferior  to  the 
dextrose  fermentation  tube. 

Other  methods  were  tried  but  they  proved  to  be  either  imprac- 
ticable, or  no  improvement  on  the  foregoing.  Eisner's  medium  was 
experimented  with,  but  it  failed  to  give  as  good  results  as  lactose 
agar,  besides  being  more  difficult  of  preparation.  The  use  of  carbol- 
iodide-broth  suggested  itself  as  a possible  means  of  differentiating 
the  troublesome  alkali  producers  Avhich  survive  the  preliminary  incu- 
bation in  carbol-broth.  The  iodide  (1  per  cent.  KI)  proved  to  add 
nothing  appreciable  to  the  inhibitive  power  of  the  carbolic  acid.  Ex- 
periments with  various  strengths  of  carbolic  acid  showed  a dilution 
of  1-1000  to  be  the  most  advantageous.  • 

The  results  of  the  work  of  which  the  foregoing  experiments  formed 
a part,  suggest  the  following  points : 

1.  When  the  dextrose  tube  method  yields  approximately  33  per 
cent,  of  CO2,  B.  coli  is  almost  invariably  present. 

2.  For  the  direct  inoculation  of  water,  the  dextrose  fermentation 
tube  is  preferable  to  the  lactose  tube. 

3.  For  polluted  waters,  incubation  in  carbol-broth  followed  by  the 
lactose  plate  gives  better  results  than  the  dextrose  tube,  while  for 
relatively  pure  waters,  the  dextrose  tube  appears  to  be  the  more 
delicate. 

4.  When  highly  polluted  waters  are  under  examination,  the  lactose 
plate  for  the  direct  inoculation  of  water  is  less  successful  than  either 
carbol-broth  or  the  dextrose  tube.  In  our  experience  this  method 
has  given  the  best  results  when  applied  to  waters  containing  a small 
number  of  other  bacteria  in  proportion  to  the  amount  of  B.  coli 
present. 

I am  indebted  to  Professor  E.  O.  Jordan,  under  whose  direction 
this  work  was  done,  for  many  helpful  suggestions  and  criticisms. 


